Urea transporter and glutamine synthetase regulation and localization in gulf toadfish gill

Transcription

1 7 The Journl of Experimentl Biology 1, 7-71 Pulished y The Compny of Biologists 9 doi:1.1/je.1575 Ure trnsporter nd glutmine synthetse regultion nd loliztion in gulf todfish gill M. Dnielle MDonld 1, *, Brnk Vulesevi, Steve F. Perry nd Ptrik J. Wlsh 1 Rosenstiel Shool of Mrine nd Atmospheri Siene, University of Mimi, Rikenker Cusewy, Mimi, FL 3319 USA nd Deprtment of Biology, University of Ottw, Ottw, Ontrio, Cnd K1N N5 *Author for orrespondene (e-mil: dmdonld@rsms.mimi.edu) Aepted 11 Deemer SUMMARY The gol of the present study ws to investigte the role of irulting ortisol nd ure in the trnsriptionl regultion of rnhil glutmine synthetse (GS), whih inorportes NH 3 into glutmte to form glutmine, nd the todfish ure trnsporter, tut, whih is involved in ure exretion ross the gill of the gulf todfish. GS (of whih there re two isoforms, LGS nd GGS) nd tut mrna expression nd tivity were mesured in todfish exposed to tretments tht would indue vrile stress responses. In ddition, the role of irulting ure in tut regultion ws investigted y infusing todfish with ure lone or in omintion with intrperitonel injetion of RU, ortiosteroid type II reeptor ntgonist. There ws.-fold upregultion in the mrna expression of the gill-speifi GS isoform (GGS) in response to ortisol infusion nd similr upregultion in the more uiquitous isoform (LGS). Furthermore, there ws signifint 1.9-fold nd 3.3-fold upregultion in the mrna expression of the todfish ure trnsporter, tut, in response to stress through rowding or exogenous ortisol loding through infusion, respetively. In ddition, tut ws found to hve ure-sensitive omponent to trnsriptionl regultion tht ws independent of irulting ortisol onentrtions. However, the hnges mesured in mrna expression of GGS, LGS nd tut did not orrespond with hnges in protein tivity. To determine the ell type(s) involved in glutmine prodution nd ure exretion, we ttempted to lolize GGS, LGS nd tut using in situ hyridiztion. This study is the first to show tht GGS nd tut expression pper to our in gill mitohondri-rih ells of todfish, suggesting tht these ells ply omined glutmine prodution nd ure exretion role, whih my hve implitions for predtor voidne. Key words: filitted diffusion, ure prodution, ure exretion, stress, ortisol, glutmine prodution, Opsnus et. INTRODUCTION The gulf todfish (Opsnus et) hs omplete ornithine-ure yle (O-UC) llowing it to exrete equl quntities of mmoni nd ure when in its nturl hitt (Hopkins et l., 1997; Hopkins et l., 1999; Brimo nd Wlsh, 5). When removed from nture nd pled under stressful onditions in the lortory (rowding, onfinement or exposure to ir or mmoni), todfish swith to exreting predominntly ure (Wlsh et l., 199; Wlsh et l., 199; Wlsh nd Millign, 1995) in pulstile mnner (1 distint events of ure exretion per h) (Wood et l., 1995; Wood et l., 1997). The trnsition to ureotelism is mrked y severl events, the first eing surge in irulting levels of the stress hormone ortisol (Wlsh et l., 199; Wlsh nd Millign, 1995; Hopkins et l., 1995). Seond is the indution of glutmine synthetse (GS) tivity in the liver, n importnt enzyme tht shuttles mmoni s glutmine into the pisine O-UC, ultimtely resulting in n inrese in ure prodution nd irulting ure levels (Wlsh et l., 199; Wlsh nd Millign, 1995; Hopkins et l., 1995). The finl two events mrking the trnsition to ureotelism involve the redution in mmoni-n exretion (Wlsh nd Millign, 1995) nd the initition of pulstile exretion of ure-n (Wood et l., 1995; Wood et l., 1997). A potentil ftor in reduing rnhil mmoni exretion is the existene of two GS isoforms in the todfish gill: uiquitous isoform tht ws initilly purified nd hrterized from the liver (hene lled LGS) (Wlsh, 199) nd is found in vrious tissues throughout the ody inluding the liver nd the gill, nd seond isoform tht, to dte, hs een found only in the gill (GGS) (Wlsh et l., 3). Although the gill is the only orgn in todfish expressing oth GS isoforms, totl GS tivity in the gill ontriutes only smll perentge (3.3%) to totl ody GS tivity, suggesting minor role in terms of whole-ody glutmine/ure prodution (Wlsh et l., 3). However, the multiple GS isoforms in the gill ould explin the redution mesured in mmoni-n exretion during the swith to ureotely (Wlsh nd Millign, 1995), with these isoforms prtiipting in the shuttling of mmoni into glutmine, gretly reduing the mount of mmoni tht rosses the gill during stressful situtions. Very little is known out the gill GS isoforms in terms of their ortisol sensitivity or, furthermore, the ell type (i.e. pvement ell or mitohondri-rih ell, MRC) (see Evns et l., 5) in whih either GS isoform is expressed. However, with respet to the ltter, it hs een speulted tht GGS my e found in pvement ells, with LGS in MRCs (Wlsh et l., 3). Extensive reserh over the pst dede hs foused on pulstile ure exretion in todfish (reviewed y Wood et l., 3). Exretion is filitted y ure trnsport protein (tut) tht shows greter thn % identity t the mino id level to mmmlin UT-A filitted diffusion ure trnsporters (Smith et l., 199; Wlsh et l., ) nd it is hypothesized tht the pulstile spet of ure exretion is due to the periodi insertion or tivtion of tut. The ellulr lotion of tut hs not een firmly estlished lthough hnges in pvement ell morphology nd inresed vesiulr trffiking within these ells during ure pulsing led to speultion tht tut nd GGS my e lolized minly in pvement ells (Lurent et l., 1).

2 tut nd GS regultion nd loliztion 75 Cirulting ortisol is n importnt regultory omponent of tut funtion tht is not ompletely understood. While it might e enefiil for the surge in ortisol mesured in onjuntion with the trnsition to ureotely to prompt n upregultion in tut trnsription s wy to filitte ure exretion ross the gill, high levels of ortisol hve onsistently een shown to inhiit tut funtion on oth n ute nd hroni level (Hopkins et l., 1995; Wood et l., 1997; Wood et l., 1; MDonld et l., ). On the ute time ourse, ure pulses pper only to our when the normlly elevted irulting ortisol onentrtions of ureoteli todfish periodilly drop, suggesting inhiition of tut funtion t high ortisol onentrtions nd the redution in ortisol ting in permissive mnner for tut tivtion (Hopkins et l., 1995; Wood et l., 1997; Wood et l., 1). On more hroni time ourse, ontinuous ortisol infusion (s wy to prevent the pre-pulse drop) results in signifint redution in the size of the ure pulse (MDonld et l., ). Comined, this evidene suggests tht elevtions in ortisol my ffet the numer of funtionl ure trnsporters, perhps through trnsriptionl or post-trnsriptionl downregultion; however, regultion of tut ould lso e n indiret result of ortisol. As mentioned previously, surge in ortisol results in the swith to ureotelism nd onsequent inrese in ure prodution y the liver. Even when lredy ureoteli, further inrese in irulting ortisol y infusion results in n dditionl inrese in plsm ure onentrtions (MDonld et l., ). While ure onentrtion hnges hve een ruled out s triggering the tul pulse event (Wood et l., 1997), there is preedent in the mmmlin literture for potentil ure effets on the trnsription of UT messge (Klein et l., 1999). Therefore, the gol of the present study ws to investigte in more detil the potentil for trnsriptionl regultion of GS, involved in the inorportion of mmoni into glutmine, nd tut, involved in ure exretion in the gill of the gulf todfish. The entrl hypothesis is tht, in response to stress, the todfish gill exhiits inresed expression of GS nd tut so s to onserve mmoni yet exrete ure. An lterntive hypothesis is tht tut mrna expression is tully downregulted y elevted plsm ortisol nd/or plsm ure levels. To test these hypotheses, GGS, LGS nd tut mrna expression nd tivity were mesured in todfish exposed to tretments to indue vrile stress responses. To determine the ell type(s) involved in glutmine prodution nd ure exretion, we ttempted to lolize GGS, LGS nd tut mrna expression using in situ hyridiztion. The role of irulting ure in tut regultion ws lso investigted y infusing todfish with ure lone or in omintion with intrperitonel injetion of RU, ortiosteroid type II reeptor ntgonist (Bertgn et l., 19; Gillrd et l., 195) tht hs een shown to prevent ortisol-indued ure exretion effets in todfish (MDonld et l., ). MATERIALS AND METHODS Experimentl nimls Gulf todfish (Opsnus et, Goode nd Ben;.7±. kg rnging from. to.191kg, N=55) were ught y ommeril shrimp fishermen in Bisyne By, FL, USA, in the spring nd summer of. The todfish were held in n outdoor tnk t the shrimpers holding fility with running se wter (mient sesonl onditions) for no longer thn h following pture, then trnsferred to the lortory. Fish were treted with dose of mlhite green (finl onentrtion.5 mg l 1 ) in formlin (15mgl 1 ) (AquVet, Hywrd, CA, USA) on the dy of trnsfer to the lortory in order to prevent infetion y the ilte Cryptoryon irritns (Stoskopf, 1993). Initilly the fish were kept in 5 l glss quri with flowing, erted sewter ( C) nd fed one weekly with previously frozen squid. Experimentl protool Series i: endogenous ortisol elevtion y rowding Unrowded todfish (N=) were kept individully in lrge, mesoosm tnks (1 m 3 ) tht simulted the nturl todfish environment, for 1 week prior to smpling. Crowded todfish (N=) were mintined together in 1l plsti ontiners for 1week prior to smpling. After the limtion period, todfish were removed from either the mesoosm or the rowding tnks, wrpped in wet pper towels nd the lood quikly smpled y udl punture. Fish were then nesthetized with lethl dose of MS- (3gl 1 ) nd gill tissue disseted. Blood smples were entrifuged t 1, g for 1min nd the plsm dented. Plsm nd gill smples were frozen immeditely in liquid nitrogen nd stored t C for no longer thn 1 month efore nlysis of plsm ure nd ortisol, gill tut nd GS mrna expression, nd GS tivity. Series ii: exogenous ortisol loding through rteril infusion Cudl rteril theteriztions nd reovery were performed s desried previously (MDonld et l., ). In protool similr to tht desried efore (MDonld et l., ), the rteril theter ws onneted to one hnnel of Gilson -hnnel peristlti pump nd fish were infused for h with isosmoti NCl (15mmoll 1 ) t n infusion rte of 3mlkg 1 h 1 ; the rte ws heked y periodi mesurement of the mss of eh individul infusion reservoir. After the h infusion with NCl, lood smple ws tken. Todfish were then seprted into two tretment groups. Sline-infused fish (men mss ± s.e.m..5±. kg, N=1) ontinued to e infused with isosmoti NCl t n infusion rte of 3mlkg 1 h 1 for seond h. Fish treted with ortisol (men mss.±.3 kg, N=1) were infused with ortisol (.19 mmol l 1 ; hydroortisone hemisuinte slt; Sigm-Aldrih Chemils, St Louis, MO, USA) in isosmoti NCl t rte of.5 μmol ortisolkg 1 h 1 for h. In oth groups, fter the seond h infusion, lood smple ws tken through the rteril theter; fish were then nesthetized with lethl dose of MS- (3gl 1 ) nd gill tissue ws disseted. Blood smples were entrifuged t 1g for 1min nd the plsm dented. Plsm nd gill smples were frozen immeditely in liquid nitrogen nd stored s desried ove. The onentrtion of infused ortisol ws hosen to rise irulting ortisol levels to pproximtely 5-fold higher thn in typil ureoteli todfish; levels whih hve een shown in previous study to result in n inhiition of pulstile ure exretion in todfish (MDonld et l., ). Series iii: exogenous ure loding through rteril infusion Arteril theter implnttion ws s desried ove. At the sme time, intrperitonel (IP) theters (Cly-Adms PE1, Frnklin Lkes, NJ, USA) filled with penut oil were inserted through smll ventrl inision nd threded pproximtely m inside the ody vity s desried y MDonld nd Wlsh (MDonld nd Wlsh, ). The fish were left to reover undistured for h, wter flow to the fish ox ws stopped, set to known volume nd n initil wter smple ws tken for the mesurement of ure onentrtion. Wter smples were ontinued s desried ove. The rteril theter ws onneted to one hnnel of Gilson - hnnel peristlti pump nd fish were infused for h with n isosmoti lod of NCl (15mmoll 1 ) t n infusion rte of 3mlkg 1 h 1. After the h infusion with NCl, lood smple ws tken nd todfish were then seprted into two tretment groups.

3 7 M. D. MDonld nd others Fish treted with ure+penut oil (men mss.±.5kg, N=1) were infused with ure (1mmoll 1 ) in isosmoti NCl t rte of 3 μmol urekg 1 h 1 for h during whih the todfish were injeted through the IP theter with.ml of penut oil strting immeditely efore the ure infusion. Injetions through the IP theter were repeted every 1h for the reminder of the experiment while fish were ontinuously infused with ure. This tretment group served s vehile nd IP injetion ontrol for fish infused with ure+ru (men mss.7±.kg, N=11). Fish in this group were infused with ure during whih they were injeted intrperitonelly with 1.5mg RU, gluoortioid reeptor ntgonist (mifepristone, 11β [-dimethylmino]phenyl-17βhydroxy-17[1-propynyl]estr-,9-dien-3-one; Sigm-Aldrih Chemils) in.1ml penut oil followed y.3ml of penut oil, strting immeditely efore the ure infusion. In oth groups, fter the seond h infusion, lood smple ws tken through the rteril theter; fish were then nesthetized with lethl dose of MS- (3gl 1 ) nd gill tissue ws disseted. Blood smples were entrifuged t 1g for 1min nd the plsm dented. Plsm nd gill smples were frozen immeditely in liquid nitrogen nd stored s desried ove. The onentrtion of infused ure ws hosen to rise irulting ure levels to pproximtely 5-fold higher thn in typil ureoteli todfish s desried previously (MDonld et l., 3). The mount of RU injeted ws hosen suh tht irulting levels of ntgonist were 1-fold greter thn irulting ortisol onentrtions. This onentrtion hd lso een shown previously to inhiit ortisol-indued redution in ure exretion in todfish (MDonld et l., ). The IP injetion of lipophili ompounds (suh s RU) in oil vehiles (i.e. penut oil, oonut oil, orn oil) medites the slow relese of these sustnes into the irultion (Vijyn nd Letherlnd, 199; Christensen et l., 1999; MDonld et l., ). Quntittive PCR Totl RNA ws isolted from whole gill within 1month of otining the smple following the protool provided with Trizol regent (Invitrogen, Crlsd, CA, USA) nd treted with DNAse to remove potentil residul genomi DNA (TuroDNA-free kit; Amion, Austin, TX, USA). DNA synthesis using rndom hexmers ws performed ording to the protool provided with the SuperSript first-strnd synthesis system for RT-PCR (Invitrogen). Quntittive PCR (qpcr) ws performed for tut, GGS nd LGS (genes of interest; GOI) using Mx multiple quntittive PCR system (Strtgene, L Joll, CA, USA) with SYBR Green. For elongtion ftor 1α (EF1α; forwrd primer 5 - GTT GGT GTC ATC AAG GCT GTT A-3, reverse primer 5 - TGA ACT CTG CCT TGA AGA TGA A-3 ), tut (forwrd primer 5 -CAT CAT CTC CCT CTT CAT CTC C-3, reverse primer 5 - GTA TCC CCA CAA GCC AAA ATA A-3 ), GGS (forwrd primer 5 -AAA CCC AGG TCA CCT ACA TCT G-3, reverse primer 5 - GCA CAC TGG GAT GAG GTA CAT A-3 ) nd LGS (forwrd primer 5 -TTG AGT AAA GCT GTC AAG AAG CA-3, reverse primer 5 -AAC CAA GTA CAT GTC GCT GTT G-3 ), primers were designed sed on pulished todfish sequenes (GenBnk ession nos AF1593, AF5331 nd AF1113 for tut, GGS nd LGS, respetively). The mount of DNA for the GOI ws expressed reltive to the mount of DNA from normlizer gene (1S for tut nd GGS, nd EF1α for LGS). Two different normlizers were used euse the undne of the normlizer gene should e similr to tht of the GOI s evidened y similr Ct vlues. The stility of normlizer gene expression with tretment ws tested y ompring normlizer gene expression in fish from different tretment groups, using DNA otined from normlized quntities of totl RNA s desried in Shmittgen nd Zkrjsek (Shmittgen nd Zkrjsek, ). To determine whether the mplifition/detetion effiienies of the GOI nd the normlizer gene were similr, stndrd urve ws generted with known quntities of DNA for the GOIs nd the normlizer gene plotted versus their Ct vlues. The stndrd urves of the GOIs nd normlizer genes in the present studies gve PCR effiienies of 1% (tut), 1% (GGS), 99.% (LGS),.% (1S) nd 7.% (EF1α). To ensure tht the mplifition of only one PCR produt ws ontriuting to the mesured Ct vlue, dissoition urve ws estlished for eh produt, whih reveled only single pek signifying only one mplified produt. No-templte ontrols were lso run to ensure tht primer onentrtions were optimized nd primer-dimers were not ontriuting to the fluoresene. To verify tht the orret produt for eh primer pir ws eing mplified, the size of the PCR produt for suset of smples ws determined using gel eletrophoresis. Gel-extrted PCR produts (with Qiex II from Qigen, Vleni, CA, USA) were then loned nd mplified in Esherihi oli ording to the protool provided with the TOPO TA loning kit for sequening using TOP1 hemilly ompetent one shot ells (Invitrogen). The plsmid DNA ws isolted (Qigen miniprep) nd the lones sequened nd identified (Genewy LLC, CA, USA). Tissue preservtion Gill filments were removed from freshly disseted gill rhes olleted from unrowded nd rowded fish s desried in series i. The filments were pled in ie-old % prformldehyde (ph7.) nd kept t C overnight. They were then trnsferred to phosphte-uffered sline (PBS) ontining 15% surose for h t C nd, finlly, trnsferred to PBS ontining 3% surose. Tissue smples were emedded in Shndon Cryomtrix emedding medium (Fisher Sientifi, Pittsurgh, PA, USA), nd setions (1 μm) were prepred using Lei CM 15 ryostt t C. Setions were pled on SuperFrost ++ (Fisher Sientifi) mirosope slides, ir dried for 3min, nd stored t C until use. Immunoytohemistry Setions were wshed in situ (3 5min) with wshing uffer ontining.1moll 1 PBS, nd.9% Triton X-1. They were then inuted for h t 37 C, in humidified hmer, with primry ntiodies diluted in the uffer: α5, mouse monolonl ntiody ginst the α 1 -suunit of hiken N + /K + -ATPse (1:1; University of Iow Hyridom Bnk). For negtive ontrols, setions were inuted with wshing uffer lking primry ntiodies. The α5 ntiody hs een used in numerous previous studies to lolize N + /K + -ATPse in fish tissues (e.g. Wilson et l., ). The slides were then wshed (3 5min) in.1moll 1 PBS. The α5 ntiody ws deteted with 1: dilution of Alex 5-oupled got ntimouse IgG (Fisher Sientifi). Slides were inuted in humid hmer for 1h t room temperture. They were then wshed (3 5min) in.1moll 1 PBS nd mounted with mounting medium (Vetor Lortories, Burlingme, MA, USA) with or without, dimidino--phenylindole (DAPI) to stin nulei. In situ hyridiztion For in situ studies, digoxigenin-leled RNA proes were prepred y in vitro trnsription using linerized plsmid DNA nd SP RNA polymerse (for ntisense) or T7 RNA polymerse (for sense). To generte homologous proe for N + /K + -ATPse, primers were

4 tut nd GS regultion nd loliztion 77 designed ginst onserved regions of the α 1 suunit. These primers (N + /K + -ATPse forwrd 5 -TAC TAC CAA GAR GCC AAG AGC T-3 ; N + /K + -ATPse reverse 5 -GTT CTG GGT CAG GGT GC- 3 ) orresponded to nuleotides 7 5 nd of the α 1 isoform of rinow trout (Onorhynhus mykiss) N + /K + -ATPse (GenBnk ession no. AY ). The resultnt 57p PCR produt ws ligted into PCR II vetor (Invitrogen) nd trnsformed into ompetent DH5α E. oli ells. Purified plsmids were sequened to onfirm tht the loned PCR produt ws homologous to N + /K + - ATPse. For tut (forwrd primer 5 -ATC ACA CGG CAC AAA GG AT-3, reverse primer 5 -ATG AAC AGC TTG GGC AAA T- 3 ), GGS (forwrd primer 5 -CGC TGT TTG GTA CAG ATG GA- 3, reverse primer 5 -GTA CGG GTC ACA GTT TGC AG-3 ) nd LGS (forwrd primer 5 -TCT TCC GGA ATG GAA CTT TG-3, reverse primer 5 -CTT CTC CTG GCC GAC ACT AC-3 ), primers were designed sed on pulished todfish sequenes. These primers were used to mplify seleted regions of full-length DNAs from previously prepred plsmids. The PCR produts (tut 73p, GGS 35p nd LGS 3p) were loned nd sequened s desried ove. Setions on slides were hydrted ( 15min) in 1 PBST (PBS with.1% Tween ). Proteinse K ( μgml 1 in 1 PBST; Gio-BRL, Ornd Islnd, NY, USA) ws used to deproteinte smples for min t room temperture. Following deproteintion, smples were fixed in % formldehyde (in PBS) for 5 min. Fixed tissues were susequently rinsed twie (1 min per wsh) with 1 PBST nd ir dried t C for 15min. Proes (pproximtely ng per retion) were dentured for 3min t 9 C in solution ontining 5 μgml 1 slmon sperm DNA, 5 μg poly(a) +, topped up to 1.5 μl with DEPC (diethyl pyroronte) H O. Proes were then quikly hilled on ie nd entrifuged (75g) for 1min. Hyridiztion uffer (1 μl of SSC, % dextrn sulfte, 5% formmide, 5 μgml 1 poly(a) +, 5 μgml 1 ssdna,.1moll 1 DTT, 5 μgml 1 trna,.5 Denhrdt s solution) ws dded to eh proe. Eh proe ws then mixed well y vortexing nd pled onto setions. Hyridiztion ws performed for h t 57 C in humid hmer. Following overnight hyridiztion, setions were wshed twie (15 min per wsh, 5 C) with SSC nd twie (15min per wsh, 5 C) with. SSC, followed y one wsh in.1 SSC for 1min t room temperture nd two wshes in.1 PBS (1min per wsh, room temperture). To detet hyridiztion, setions were inuted for 1h t room temperture with 1% got serum, mgml 1 BSA in.1moll 1 PBS with.3% Triton X-1, followed y overnight inution t C in nti-digoxigenin ntiody onjugted to lkline phosphtse (1:1 dilution; Rohe Moleulr Biohemils, Temeul, CA, USA). Slides were wshed t room temperture in.1moll 1 PB for 15min nd then riefly rinsed in wter. The slides were next wshed twie (5min per wsh) in olortion uffer (1mmoll 1 Tris ph9.5, 5mmoll 1 MgCl, 1mmoll 1 NCl,.1% Tween ). Nitrolue tetrzolium (NBT) nd single 5-romoresyl-3-indolyl phosphte (BCIP) tlet (Sigm-Aldrih Chemils) were dissolved in 1ml of H O nd lyered over the setions. Color ws llowed to develop in humid hmer t room temperture for t lest h or until stisftory olortion ws oserved. The slides were then wshed twie with.1moll 1 PBS (15min per wsh). Coverslips were pled on the slides using % glyerol s mounting medium. One prepred, ll speimens were oserved nd photogrphed using Zeiss Axiophot mirosope (Zeiss, Jen, Germny) equipped with Hmmtsu C595 hilled CCD mer, using Metmorph imging softwre.1 (Moleulr Devies, Dowingtown, PA, USA). Assys Ure onentrtions in plsm nd wter were mesured using the dietyl monoxime method of Rhmtullh nd Boyde (Rhmtullh nd Boyde, 19) with pproprite djustments of regent strength for the different ure onentrtion rnges in wter nd lood plsm. Totl ure exretion ws mesured nd lulted s desried previously (MDonld et l., ). GS tivity in gill tissue ws mesured using the trnsferse ssy s desried y Wlsh (Wlsh, 199). Plsm ortisol onentrtions were mesured using ommeril 15 I rdioimmunossy kit (MP Biomedil, Solon, OH, USA) with stndrds diluted to the sme protein rnge s todfish plsm. Sttistis Dt re reported s mens ± 1s.e.m. (N=numer of fish). The signifine of differenes etween mens ws evluted using Student s unpired two-tiled t-test (P<.5). When dt were still not normlly distriuted upon log trnsformtion, Mnn Whitney rnk sum test ws used. RESULTS Plsm ortisol onentrtions were lowest in fish tht were unrowded in the mesoosm tnks (Fig. 1A). In omprison, fish tht were kept t high density for 1week hd ortisol onentrtions tht were more thn 1-fold higher (Fig. 1A). Sline infusion resulted in ortisol onentrtions tht were signifintly higher thn those in unrowded todfish ut signifintly lower thn those in fish tht were infused with ortisol, whih hd onentrtions tht exeeded those of unrowded fish y pproximtely 17-fold (Fig. 1A). Fish infused with ure or ure+ru hd plsm ortisol onentrtions tht were signifintly higher thn those of unrowded fish ut were similr to those of rowded nd slineinfused fish (Fig. 1A). However, ure-infused fish with or without RU tretment hd signifintly lower ortisol onentrtions thn fish tht were ortisol infused. Like irulting ortisol onentrtions, plsm ure onentrtions were lso lowest in unrowded todfish (Fig. 1B). Ure onentrtions were signifintly inresed in fish tht were sline infused, ut fish tht were just rowded were not different from unrowded or sline-infused fish. Fish tht were infused with ortisol hd even higher irulting ure onentrtions thn those infused with sline nd further inrese ws mesured in fish tht were ure infused either with or without RU tretment (Fig. 1B). Totl GS tivity ws signifintly higher in ll tretment groups ompred with tht in the unrowded, ontrol fish, with the gretest elevtion eing mesured in the rowded todfish (Fig. A). The reltive expression of GGS mrna did not show the sme level of sensitivity to tretment s GS tivity (Fig. B). Speifilly, GGS mrna expression in rowded, sline-infused or ure-infused ±RU fish ws not signifintly different from tht mesured in unrowded fish (Fig. B). However,.-fold elevtion in GGS expression ws mesured in fish tht were infused with ortisol (Fig. B). The mrna expression of the LGS isoform showed slightly more sensitivity to tretment thn tht of the GGS isoform. Similr to GGS, rowding hd no effet on LGS mrna expression; however, sline infusion resulted in signifint.7-fold inrese in LGS mrna expression, whih remined elevted in the other tretments (Fig. C). Nonetheless, rough omprison of normlized GGS nd LGS trnsript undne indites tht the GGS isoform ws pproximtely -fold more undnt in the gill under ll onditions tested thn the LGS isoform (dt not shown).

5 7 M. D. MDonld nd others Plsm [ortisol] (ng ml 1 ) Plsm [ure] (mmol l 1 ) A B,d,, The reltive expression of tut mrna showed 1.9-fold nd 1.7-fold elevtion in rowded nd sline-infused fish, respetively, ompred with fish tht were held in unrowded onditions (Fig. 3). Fish tht were ortisol infused hd tut mrna levels tht were 3.3- fold higher thn those of unrowded fish nd signifintly greter thn those of oth rowded nd sline-infused fish. Interestingly,.-fold inrese ws seen in ure-infused fish ompred with unrowded fish, whih ws redued to 3.-fold inrese in fish tht were treted with ure+ru (Fig. 3). Despite hving higher levels of tut mrna expression, fish tht were infused with ortisol hd signifintly lower rtes of ure exretion thn fish infused with sline lone (Fig. A f. Fig. 3). Along the sme lines, fish infused with ure+ru tretment showed n elevtion in ure exretion ompred with the h ontrol NCl infusion tht ws similr to tht in fish infused with ure lone, despite RU-treted fish hving lower tut mrna expression levels thn fish infused with ure lone (Fig.B f. Fig.3). Bsed on the results of immunoytohemistry nd in situ hyridiztion (Fig.5), it ws pprent tht the MRCs (identified on the sis of N + /K + -ATPse enrihment) of the gill epithelium typilly were sttered long the lmelle (Fig. 5A,B). Notly, in single fish, it ws oserved tht the MRCs were speifilly lolized to the interlmellr regions within the filment epithelium. There ws no ovious effet of holding onditions (rowded versus non-rowded) on the pttern of MRC distriution. The distriution pttern of GGS nd tut mrna ws similr to tht of N + /K + - ATPse, suggesting their exlusive loliztion to the MRCs (Fig. 5C,D). Positive stining for LGS ws undetetle. d, d Unrowded Crowded Sline infused Cortisol infused Ure infused Ure+RU, Series i Series ii Series iii Fig. 1. (A) Plsm ortisol onentrtions nd (B) plsm ure onentrtions in todfish tht were reltively unstressed (unrowded) ompred with those under different experimentl tretments. Vlues re mens + 1 s.e.m. Different letters depited signifint differene, P<.5. d Glutmine synthetse tivity (µmol g 1 min 1 ) Reltive GGS mrna expression Reltive LGS mrna expression A B C,,, Series i Series ii Series iii Unrowded Crowded Sline infused Cortisol infused Ure infused Ure+RU Fig.. (A) Totl glutmine synthetse (GS) tivity, (B) reltive mrna expression of the gill-speifi GS isoform (GGS) nd (C) reltive mrna expression of the uiquitous isoform (LGS). Notly, tretments tht resulted in hnges in enzyme tivity do not orrespond to mrna expression hnges in either GS isoform. Vlues re mens + 1 s.e.m. Different letters depit signifint differene, P<.5. DISCUSSION The present study is the first to demonstrte tht todfish experiene n upregultion in the mrna expression of rnhil GS (oth GGS nd LGS isoforms) nd the todfish ure trnsporter, tut, in response to rowding or exogenous ortisol loding through infusion. Furthermore, tut ppers to hve ure-sensitive omponent to trnsriptionl regultion s well. GGS nd tut expression pper to our in gill MRCs, suggesting tht these ells ply omined glutmine prodution/mmoni sequestrtion nd ure exretion role, while LGS ws found in low trnsript levels in the gill y qpcr nd ws undetetle with in situ hyridiztion.

6 tut nd GS regultion nd loliztion 79 Reltive tut mrna expression Unrowded Crowded Sline infused Cortisol infused Ure infused Ure+RU Series i Series ii Series iii Fig. 3. Reltive tut mrna expression in gill of todfish tht re unstressed (unrowded) ompred to those under different experimentl tretments. In ddition to elevtions in tut mrna expression oserved in ortisol-infused fish, the gretest elevtion in expression ws mesured in fish tht were infused with ure. Vlues re mens + 1 s.e.m. Different letters depited signifint differene, P<.5. d Dily ure exretion (µmol N kg 1 ) A Sline infusion Tretment Sline infusion B *, *, Sline infusion Tretment Cortisol infusion Ure+RU Ure infusion Gill GS tivity nd mrna expression An endogenous elevtion in irulting ortisol levels in response to rowding under lortory onditions hs een shown to result in signifint inrese in the tivity nd mrna expression of GS in the liver of todfish (Hopkins et l., 1995; Wlsh et l., 3). In orrespondene with these doumented hnges in ure prodution y the liver, todfish in the present study showed signifint elevtion in the totl GS tivity within the gill ( mesure tht inludes the tivity of oth LGS nd GGS isoforms). Sensitivity of non-hepti GS to ortisol hs een demonstrted in tilpi gstrointestinl trt, stomh nd musle (Mommsen et l., 3) s well s in mmmlin stroytes (O Bnion et l., 199) nd intestine (Srntos et l., 199). In todfish, the inrese in gill totl GS tivity in response to rowding lone is in ontrst to previous findings, whih showed no hnge (Wlsh et l., 3); however, the inonsisteny etween the two studies ould e explined y protool differenes; todfish in the previous study were only rowded for h (nd levels of stress were not estimted y mesurement of ortisol levels) ompred with the present 1 week rowding protool. While rowding resulted in n inrese in the totl GS tivity in the gill, there ws no orresponding inrese in the mrna expression of either LGS or GGS with rowding. Indution of the GS enzyme without hnges in trnsription hs een mesured in the mmmlin jejunum; in this se it ws postulted tht gluoortioids inresed GS levels y elerting protein trnsltion (Srntos et l., 199). In ontrst to fish tht were simply rowded, n inrese in the trnsription of oth LGS nd GGS isoforms ws mesured in fish tht were infused with sline or ortisol, respetively. This result is unlikely to e explined y mesured differenes in plsm ortisol onentrtions mongst the three groups (the rowded nd ortisoltreted fish hd similr levels of irulting ortisol). The upregultion of LGS mrna expression ould e in response to the volume loding experiened y infused fish; however, this would not explin the upregultion mesured in GGS, whih ppers only to e sensitive to ortisol infusion. Another differene etween rowded, sline-infused nd ortisol-infused fish is the stressor involved; the soil stress experiened y rowded fish is very intense nd, in ddition to ortisol elevtion, ould result in hnges Time (h) Fig.. (A) Dily exretion of ure in todfish tht were sline infused for n initil h ontrol period nd were then swithed to eing infused either with sline or with ortisol for susequent h. A signifint derese in exretion ws mesured in ortisol-treted fish ompred with their ure exretion vlues mesured during the ontrol period nd ompred with sline-infused fish t the sme time point. (B) Dily exretion of ure in todfish tht were sline infused for n initil h ontrol period nd were then swithed to eing infused with either ure lone or ure+ru. Both groups showed signifint elevtion in ure exretion ompred with the initil sline ontrol period; however, the tretments were not signifintly different from one nother. Vlues re mens ± 1 s.e.m.; *P<.5, signifintly different from orresponding tretment group; P<.5, signifintly different from ontrol infusion period. in mny other physiologil prmeters tht my interfere with the hnges in GGS gene expression mesured in fish infused with ortisol lone (Slomn et l., 5). It lso nnot e ruled out tht the vried exposure to different ute stressors experiened y slinend ortisol-infused fish (i.e. surgery) my hve resulted in periods of muh greter ortisol levels tht were not ptured y the single post-infusion lood smple. Nevertheless, the pttern of trnsriptionl upregultion mesured in GGS nd LGS suggests tht more hroni stressor my e required to inrese GGS trnsription thn is neessry for the upregultion of LGS. Interestingly, totl GS enzyme tivity ws not different etween sline- nd ortisol-infused fish, whih is refleted in similr rnhil mmoni exretion rtes etween the two groups (dt not shown); however, this my e onsequene of reltively smll trnsriptionl hnges not trnslting to detetle hnges in protein tivity. The signifintly higher GGS ompred with LGS trnsript levels suggests tht GGS proly mkes up greter proportion of the totl GS tivity of the gill, providing funtionl signifine to this gill-speifi isoform nd putting into ontext the differenes mesured in the pprent ortisol sensitivity of the two isoforms. Wlsh nd ollegues (Wlsh et l., 3) determined tht the

7 71 M. D. MDonld nd others ellulr omprtmenttion of GGS differs from tht of LGS, euse GGS is missing the mitohondril leder sequene tht would trget it to the mitrohondril omprtment, nd it hd n exlusively solule/ytosoli distriution, potentilly inresing its diret ontt with mmoni. The demonstrted ytosoli lotion of GGS omined with the higher trnsript levels of GGS ompred with those of LGS in the gill supports n mmoni-trpping funtion of the gill itself tht differs from the funtion of GS in other orgns. Tht higher ortisol onentrtions re required to inrese GGS trnsription thn re neessry for the upregultion of LGS suggests tht glutmine prodution my our seondry to the inrese in ure prodution in response to stress y liver-ound LGS, s the pttern of mrna expression mesured in LGS proly reflets wht is going on in the liver. Gill-ound LGS proly does not ply mjor role in gill glutmine prodution s indited y the low trnsript levels nd lk of LGS signl using in situ hyridiztion. tut tivity nd mrna expression The findings of the present study suggest tht oth ortisol nd ure hve potentil regultory effets on todfish gill tut tivity nd mrna expression. Previous studies investigting the role of ortisol in the regultion of todfish pulstile ure exretion hve onsistently demonstrted n inhiitory effet of oth ute nd hroni elevtions of ortisol on tut funtion (Hopkins et l., 1995; Wood et l., 1997; Wood et l., 1; MDonld et l., ). These pst results were further supported y the present study, in whih exogenous ortisol loding through infusion resulted in signifint derese in ure exretion, suggesting potentil downregultion of tut mrna expression or funtion. A derese in mrna undne in response to gluoortioid tretment ws mesured in severl mmmlin filitted diffusion ure trnsporters, nmely UT-A1, UT-A3 nd UT-A3 found in the inner medullry olleting dut (IMCD); gluoortioids suppressing the tivity vi the promoter region responsile for trnsription (Knepper et l., 1975; Nruse et l., 1997; Peng et l., ). However, Peng nd ollegues (Peng et l., ) did not find ny hnge in the trnsription of UT-A in response to gluoortioids, whih is the mmmlin isoform tht most losely resemles todfish tut (Wlsh et l., ). In ontrst to the hypothesized downregultion or potentil insensitivity mesured y Peng nd ollegues (Peng et l., ), the mrna undne of tut in fish with either endogenous (through rowding) or exogenous (through infusion) elevtions in ortisol ws signifintly inresed ompred with tht of unrowded fish, reveling ler distintion etween the trnsription of tut nd the pity of the fish to exrete ure, mesure of tut protein funtion. An inrese in tut mrna undne in ssoition with the surge in ortisol during the trnsition to ureotely would e dptive for timing the inrese in ure prodution with n inresed ility to exrete ure ross the gill. Wht hs eome pprent, however, is tht ortisol my hve two roles in tut regultion (Hopkins et l., 1995; Wood et l., 1997; Wood et l., 1; MDonld et l., ). It ppers tht hroni elevtion in irulting ortisol serves to inrese tut mrna expression, whih my in ft llow more ure trnsporter to e trnslted. However, elevted ortisol levels mesured in ureoteli todfish lso pper to prevent ure from eing exreted. When irulting ortisol onentrtions periodilly drop, pulse of ure ours, suggesting tht the ortisol drop is permissive to the post-trnsriptionl modifition of tut (Hopkins et l., 1995; Wood et l., 1997; Wood et l., 1). Without the periodi, nturl drop in ortisol, s oserved in todfish infused Fig. 5. The pttern of N + /K + -ATPse (A,B), GGS (C) nd tut (D) loliztion in the todfish gill whether y in situ hyridiztion (A,C,D) or immunoytohemistry (B), demonstrting N + /K + -ATPse-positive mitohondri-rih ells (MRCs) sttered long the lmelle (A,B). The insets in A nd B represent no-proe negtive ontrol nd negtive ontrol in whih primry ntiody ws omitted, respetively. GGS (C) nd tut mrna (D) showed similr pttern of stining to N + /K + -ATPse, sttered long the lmelle nd within the interlmellr regions. The sle rs indite 5 μm. with ortisol, the newly trnsried tuts might never e utilized. Alterntively, mye the inrese in tut mrna undne is simply (filed) ttempt to offset n inresed degrdtion of tut protein tht my e ourring in response to elevted ortisol onentrtions, s suggested y Kong nd ollegues (Kong et l., ) in the se of todfish rmoyl phosphte synthetse III. Then gin, there ould e seond ure trnsporter in the gill, s evidened y Wlsh nd ollegues (Wlsh et l., ) using northern nlysis, tht responds to ortisol with downregultion in trnsription whih overwhelms the upregultion mesured in tut, resulting in the mesured overll derese in ure exretion. The differing trnsriptionl responses of tut nd the losely relted mmmlin UT-A isoform to ortisol egged the question of whether ortisol ws ting diretly on tut or whether its pprent sensitivity to ortisol ws insted due to the elevtion in irulting ure onentrtions tht is often mesured in stressed todfish nd other teleosts (Vijyn et l., 199; MDonld nd Wood, ). An upregultion of mmmlin UT-A protein in response to uremi, pthologil ondition resulting in higher irulting ure onentrtions, ws originlly doumented in rts sujeted to nephretomy (Klein et l., 1999). However, lter study y Klein nd ollegues (Klein et l., ) determined tht it ws the idosis tht ourred in response to nephretomy tht diretly resulted in the inrese in UT-A undne nd not the elevtion in ure onentrtions tht ws yprodut of the idosis (Klein et l., ). In the present study, there did pper to e ure-sensitive omponent to tut mrna expression; however, lood ph in todfish infused with ure ws not mesured nd thus it nnot e onlusively ruled out tht idosis ws ontriuting ftor.

8 tut nd GS regultion nd loliztion 711 An inresed expression of UT-A trnsporters hs lso een shown to our in response to the hydrtion stte of the niml (Smith et l., 1995), with the inrese in trnsription resulting from the tivtion of the UT-A promoter y AMP-dependent pthwys (Nkym et l., 1). Chnges in hydrtion sttus sed on the volume loding experiened y oth ortisol-infused nd ureinfused fish would not explin the signifint differene in tut trnsription mesured etween these two groups. Furthermore, signifint differene in tut mrna expression is not oserved etween rowded todfish nd those tht re sline infused, two groups tht hve similr plsm ortisol nd ure levels ut differ in tht the ltter is volume loded. Thus, it ppers tht the hydrtion sttus of todfish is proly not regultory omponent of tut trnsription. In situ hyridiztion nd ololiztion of GGS nd tut Beuse of the lk of mitohondril leder sequene in GGS, Wlsh nd ollegues (Wlsh et l., 3) speulted tht the gill nd uiquitous forms of GS might e expressed in different ell types; GGS in pvement ells nd LGS in MRCs. In mrked ontrst to their speultion, GGS showed similr pttern of stining to N + /K + -ATPse, whih is expressed in MRCs, while we were unle to determine the lotion of LGS, proly due to its mrkedly lower trnsript undne s demonstrted y qpcr mesurements. Similr to GGS, nd in ontrst to speultion y Lurent nd ollegues (Lurent et l., 1), tut lso showed similr pttern of stining to N + /K + -ATPse, suggesting MRC loliztion. Chnges in pvement ell morphology nd inresed vesiulr trffiking during ure pulsing suggested tht tut my e found in gill pvement ells (Lurent et l., 1). However, the present findings re in greement with immunohistohemistry on lrgely mmonioteli speies, the Jpnese eel (Anguill jponi) ure trnsporter (eut), whih ws lolized to the solterl memrne of MRCs (Mistry et l., 1). Thus, the todfish MRCs express oth GGS nd tut nd these ells proly hve omined funtion to redue mmoni exretion y produing glutmine while t the sme time exreting ure in times of stress. Interestingly, the elortion of the pvement ells suggests exretion of n eletrondense mteril to the pil surfe vi vesiles (Lurent et l., 1) nd, in the light of reent ehviorl studies in todfish (Slomn et l., 5; Brimo nd Wlsh, ), it would e of interest to exmine these mterils for moleules involved in ommunition tht might ompny ure nd mmoni exretion s regulted y the hloride ells. Reent evidene hs outlined n importnt role for the omined redution of mmoni exretion nd elevtion in ure exretion in todfish survivl. Todfish hve een shown to exrete omintion of ure nd mmoni in the wild (Hopkins et l., 1997; Hopkins et l., 1999). A reent study hs reveled mmoni wste to e n importnt hemil ttrtnt in the quti environment whih eomes undetetle if exreted in omintion with ure (Brimo nd Wlsh, ). Hving the omined ontrol to upregulte ure-n prodution in the liver, nd upregulte ure-n exretion ross the gill while deresing mmoni-n exretion ross the gill when under stressful onditions ensures tht the fish will e exreting the pproprite mix of mmoni:ure s predtorvoidne tti. When under onditions of very high stress, suh s in lortory environment, evidene shows tht fish lmost shut down mmoni exretion entirely, eoming predominntly ureoteli (Wlsh, 1997; Wood et l., 1995; Wood et l., 1997, Wood et l., 199; Wood et l., 1). In terms of predtor voidne where nturl nlog to the high stress lortory model might e fish under repeted ttk y predtors, this full ureotely response is not disdvntgeous; Brimo nd Wlsh (Brimo nd Wlsh, ) did not find signifint differene in the ility of mmoni+ure or ure lone to ttrt/void predtors. M.D.M. is supported y NSF (IOS-559), S.F.P. is supported y n NSERC Disovery Grnt nd P.J.W. is supported y n NSERC Disovery Grnt, the Cnd Reserh Chir Progrm nd the Cnd Foundtion for Innovtion. Speil thnks to Edwrd M. Mger for tehnil ssistne with qpcr nd the use of his 1S primers. Our grtitude lso goes to the shrimp fishermen t Jimoʼs nd Mr Ry Hurley for their supply of todfish. REFERENCES Brimo, J. F. nd Wlsh, P. J. (5). The effets of ute nd hroni mmoni exposure during erly life stges of the gulf todfish, Opsnus et. Aqut. Toxiol. 75, Brimo, J. F. nd Wlsh, P. J. (). Use of ure s hemosensory loking moleule y ony fish. J. Exp. Biol. 9, 5-1. Bertgn, X., Bertgn, C., Luton, J., Husson, J. nd Gird, F. (19). The new steroid nlog RU inhiits gluoortioid tion in mn. J. Clin. Endorinol. Met. 59, 5-. Christensen, L. J., Krsgrd, B. nd Bjerregrd, P. (1999). The effet of - nonylphenol on the synthesis of vitellogenin in the flounder Ptihthys flesus. Aqut. Toxiol., Evns, D. H., Piermrini, P. M. nd Choe, K. P. (5). The multifuntionl fish gill: dominnt site of gs exhnge, osmoregultion, id-se regultion nd exretion of nitrogen wste. Physiol. Rev. 5, Gillrd, R. C., Poffet, D., Riondel, A. M. nd Surt, J. (195). RU inhiits peripherl effets of gluoortioids in humns. J. Clin. Endorinol. Met. 57, 3-5. Hopkins, T. E., Wood, C. M. nd Wlsh, P. J. (1995). Intertions of ortisol nd nitrogen metolism in the ureogeni gulf todfish Opsnus et. J. Exp. Biol. 19, Hopkins, T. E., Serfy, J. E. nd Wlsh, P. J. (1997). Field studies on the ureogeni gulf todfish, in sutropil y.. Nitrogen exretion physiology. J. Fish Biol. 5, Hopkins, T. E., Wood, C. M. nd Wlsh, P. J. (1999). Nitrogen metolism nd exretion in n intertidl popultion of the gulf todfish, Opsnus et. Mr. Freshwter Behv. Physiol. 33, 1-3. Klein, J. D., Timmer, R. T., Rouillrd, P., Biley, J. L. nd Snds, J. M. (1999). UT- A ure trnsporter portein expressed in liver: upregultion y uremi. J. Am. So. Nephrol. 1, 7-3. Klein, J. D., Rouillrd, P., Roerts, B. R. nd Snds, J. M. (). Aidosis medites the upregultion of UT-Aprotein in livers from uremi rts. J. Am. So. Nephrol. 13, Knepper, M. A., Dnielson, R. A., Sidel, G. M. nd Johnston, K. H. (1975). Effets of dietry protein restrition nd gluoortioid dministrtion on ure exretion in rts. Kidney Int., Kong, H., Khtpitiy, N., Kingsley, K., Slo, W. L., Anderson, P. M., Wng, Y. S. nd Wlsh, P. J. (). Indution of rmoyl phosphte synthetse III nd glutmine synthetse mrna during onfinement stress in gulf todfish (Opsnus et). J. Exp. Biol. 3, Lurent, P., Wood, C. M., Wng, Y., Perry, S. F., Gilmour, K. M., Prt, P., Chevlier, C., West, M. nd Wlsh, P. J. (1). Intrellulr vesiulr trffiking in the gill epithelium of ure-exreting fish. Cell Tissue Res. 33, MDonld, M. D. nd Wlsh, P. J. (). 5-HT A-like reeptors re involved in triggering pulstile ure exretion in the gulf todfish, Opsnus et. J. Exp. Biol. 7, 3-. MDonld, M. D. nd Wood, C. M. (). The effet of hroni ortisol elevtion on ure metolism nd exretion in the rinow trout (Onorhynhus mykiss). J. Comp. Physiol. 17B, MDonld, M. D., Wood, C. M., Wng, Y. nd Wlsh, P. J. (). Differentil rnhil nd renl hndling of ure, etmide nd thioure in the gulf todfish, Opsnus et: evidene for two trnsporters. J. Exp. Biol. 3, MDonld, M. D., Grosell, M., Wood, C. M. nd Wlsh, P. J. (3). Brnhil nd renl hndling of ure in the gulf todfish, Opsnus et: the effet of exogenous ure loding. Comp. Biohem. Physiol. 13A, MDonld, M. D., Wood, C. M., Grosell, M. nd Wlsh, P. J. (). Gluoortioid reeptors re involved in the regultion of pulstile ure exretion in todfish. J. Comp. Physiol. 17B, 9-5. Mistry, A. C., Hond, S., Hirt, T., Kto, A. nd Hirose, S. (1). Eel ure trnsporter is lolized to hloride ells nd is slinity dependent. Am. J. Physiol. 1, R159-R1. Mommsen, T. P., Busy, E. R., von Shlurg, K. R., Evns, J. C., Oshoff, H. L. nd Elliott, M. E. (3). Glutmine synthetse in tilpi gstrointestinl trt: zontion, DNA nd indution y ortisol. J. Comp. Physiol. 173, Nkym, Y., Nruse, M., Krkshin, A., Peng, T., Snds, J. M. nd Bgnso, S. M. (1). Cloning of the rt SLC1A gene nd genomi orgniztion of the UT- A ure trnsporter. Biohim. Biophys. At 151, 19-. Nruse, M., Klein, J. D., Ashkr, Z. M., Jos, J. D. nd Snds, J. M. (1997). Gluoortioids downregulte the vsopressin-regulted ure trnsporter in rt terminl inner medullry olleting duts. J. Am. So. Nephrol., OʼBnion, M. K., Young, D. A. nd Bohn, M. C. (199). Cortiosterone-responsive mrnas in primry rt stroytes. Mol. Brin Res., 57-. Peng, T., Snds, J. M. nd Bgnso, S. M. (). Gluoortioids inhiit trnsription nd expression of the UT-A ure trnsporter gene. Am. J. Physiol., F53-F5.

9 71 M. D. MDonld nd others Rhmtullh, M. nd Boyde, T. R. (19). Improvements in the determintion of ure using dietyl monoxime: methods with nd without deproteintion. Clin. Chim. At 17, 3-9. Srntos, P., Chkrrti, R. nd Copelnd, E. M. (199). Dexmethsone inreses jejunl glutmine synthetse expression vi trnsltionl regultion. Am. J. Surg. 17, -13. Shmittgen, T. D. nd Zkrjsek, B. A. (). Effet of experimentl tretment on housekeeping gene expression: vlidtion y rel-time, quntittive RT-PCR. J. Biohem. Biophys. Methods, 9-1. Slomn, K. A., MDonld, M. D., Brimo, J. F., Lepge, O., Winerg, S., Wood, C. M. nd Wlsh, P. J. (5). Does pulstile ure exretion serve s soil signl in the gulf todfish, Opsnus et? Physiol. Biohem. Zool. 7, Smith, C. P., Heitz, M. J., Wood, C. M. nd Wlsh, P. J. (199). Moleulr identifition of gulf todfish (Opsnus et) ure trnsporter. J. Physiol. 511, 33P. Stoskopf, M. K. (1993). Fish Mediine. Phildelphi, PA: W.B. Sunders. Vijyn, M. M. nd Letherlnd, J. F. (199). Cortisol-indued hnges in plsm gluose, protein, nd thyroid hormone levels, nd liver glyogen ontent of oho slmon (Onorhynhus kisuth Wlum). Cn. J. Zool. 7, Vijyn, M. M., Mommsen, T. P., Glemet, H. C. nd Moon, T. W. (199). Metoli effets of ortisol tretment in mrine teleost, the se rven. J. Exp. Biol. 199, Wlsh, P. nd Millign, C. (1995). Effets of feeding nd onfinement on nitrogen metolism nd exretion in the gulf todfish Opsnus et. J. Exp. Biol. 19, Wlsh, P. J. (199). Purifition nd properties of hepti glutmine synthetses from the ureoteli gulf todfish, Opsnus et. Comp. Biohem. Physiol. 115B, Wlsh, P. J. (1997). Evolution nd regultion of ure synthesis nd ureotely in (trhoidid) fishes. Annu. Rev. Physiol. 59, Wlsh, P. J., Dnult, E. M. nd Mommsen, T. P. (199). Vrition in ure exretion in the gulf todfish, Opsnus et. Mr. Biol. 1, Wlsh, P. J., Tuker, B. C. nd Hopkins, T. E. (199). Effets of onfinement/rowding on ureogenesis in the gulf todfish, Opsnus et. J. Exp. Biol. 191, Wlsh, P. J., Heitz, M. J., Cmpell, C. E., Cooper, G. J., Medin, M., Wng, Y. S., Goss, G. G., Vinek, V., Wood, C. M. nd Smith, C. P. (). Moleulr hrteriztion of ure trnsporter in the gill of the gulf todfish (Opsnus et). J. Exp. Biol. 3, Wlsh, P. J., Myer, G. D., Medin, M., Bernstein, M. L., Brimo, J. F. nd Mommsen, T. P. (3). A seond glutmine synthetse gene with expression in the gills of the gulf todfish (Opsnus et). J. Exp. Biol., Wilson, J. M., Lurent, P., Tufts, B. L., Benos, D. J., Donowitz, M., Vogl, A. W. nd Rndll, D. J. (). NCl uptke y the rnhil epithelium in freshwter teleost fish: n immunologil pproh to ion-trnsport protein loliztion. J. Exp. Biol. 3, Wood, C., Hopkins, T., Hogstrnd, C. nd Wlsh, P. (1995). Pulstile ure exretion in the uregeni todfish Opsnus et: n nlysis of rtes nd routes. J. Exp. Biol. 19, Wood, C. M., Hopkins, T. E. nd Wlsh, P. J. (1997). Pulstile ure exretion in the todfish (Opsnus et) is due to pulstile exretion mehnism, not pulstile prodution mehnism. J. Exp. Biol., Wood, C. M., Gilmour, K. M., Perry, S. F., Prt, P. nd Wlsh, P. J. (199). Pulstile ure exretion in gulf todfish (Opsnus et): evidene for tivtion of speifi filitted diffusion trnsport system. J. Exp. Biol. 1, Wood, C. M., Wrne, J. M., Wng, Y., MDonld, M. D., Blment, R. J., Lurent, P. nd Wlsh, P. J. (1). Do irulting plsm AVT nd/or ortisol levels ontrol pulstile ure exretion in the gulf todfish (Opsnus et)? Comp. Biohem. Physiol. 19A, Wood, C. M., MDonld, M. D., Sundin, L., Lurent, P. nd Wlsh, P. J. (3). Pulstile ure exretion in the gulf todfish: mehnisms nd ontrols. Comp. Biohem. Physiol. 13B, 7-.